本文選題：慢性缺氧 + 心室重構(gòu)　； 參考：《昆明醫(yī)科大學(xué)》2017年碩士論文

【摘要】：[目的]慢性缺氧是導(dǎo)致心室重構(gòu)的主要因素之一。心室重構(gòu)是導(dǎo)致各種心臟病發(fā)生心衰、心律失常的重要的病理機(jī)制,也是心臟老化的標(biāo)志。近年來國內(nèi)外研究包括本課題組前期實驗研究發(fā)現(xiàn)BMSCs (骨髓間充質(zhì)干細(xì)胞)移植可有效改善心肌細(xì)胞凋亡及心肌纖維化,BMSCs移植治療可望成為改善心室重構(gòu)的可行性治療手段之一。但BMSCs改善心室重構(gòu)的作用機(jī)制尚不甚清楚。PNUTS(Serine/threonine protein phosphatase 1 regulation 10; PPPIR10;絲氨酸/蘇氨酸蛋白磷酸酶1調(diào)節(jié)亞基10)是蛋白磷酸酶1 ( PP1)的調(diào)節(jié)亞基,其主要有抗細(xì)胞凋亡、促進(jìn)急性心肌梗死后心功能恢復(fù)的作用,近來有研究表明BMSCs中可表達(dá)PNUTS,但是BMSCs是否通過PNUTS改善慢性缺氧導(dǎo)致的心室重構(gòu)以及PNUTS和慢心缺氧導(dǎo)致的心室重構(gòu)的相關(guān)性未見報道。故本課題建立SD大鼠慢性缺氧心室重構(gòu)模型,并用BMSCs治療各缺氧大鼠模型。檢測慢性缺氧心室重構(gòu)模型及BMSCs治療后大鼠的心室miR-34a、pnuts mRNA表達(dá)量、PNUTS濃度,以及血清和心肌組織中的PⅠCP、PⅢNP濃度。探討PNUTS在慢性缺氧所致的心室重構(gòu)及BMSCs改善慢心缺氧導(dǎo)致的心室重構(gòu)中的作用。[方法]45只大鼠隨機(jī)分為9組,每組5只,分別為:A組(對照組)、B組(缺氧7天)、C組(缺氧14天)、D組(缺氧21天)、E組(缺氧28天)、B'組(缺氧7天后腹腔注射濃度為5×106/mL的干細(xì)胞1mL,治療4周)、C'組(缺氧14天后腹腔注射濃度為5×106/mL的干細(xì)胞1mL,治療4周)、D'組(缺氧21天后腹腔注射濃度為5×106/mL的干細(xì)胞1mL,治療4周)、E'組(缺氧28天后腹腔注射濃度為5×106/mL的干細(xì)胞1mL,治療4周),運用全自動缺氧實驗裝置建立缺氧組模型,運用干細(xì)胞治療4周后稱取大鼠體重,按0.3mL/100g的量腹腔注射10%水合氯醛麻醉大鼠并進(jìn)行解剖,打開腹腔,于腹主動脈采血,離心后取血清;打開胸腔取心臟制作病理切片,HE染色觀察各缺氧組及治療組大鼠心肌組織的細(xì)胞形態(tài)、水腫、肥大、變性、壞死及炎性細(xì)胞浸潤情況等病理性改變;Masson染色觀察各缺氧組及治療組大鼠心肌組織的心肌膠原分布及含量,計算膠原容積分?jǐn)?shù)(CVF);熒光Tunel檢測各缺氧組及治療組大鼠心肌組織中心肌細(xì)胞凋亡情況,計算細(xì)胞凋亡率;運用qPCR檢測各缺氧組、治療組大鼠心肌組織及BMSCs中miR-34a表達(dá)量、pnuts mRNA表達(dá)量;運用ELISA檢測各缺氧組和治療組大鼠血清中PⅠCP、PⅢNP濃度,心肌組織中PⅠCP、PⅢNP濃度,PNUTS濃度,BMSCs中PNUTS濃度;分析miR-34a表達(dá)量、pnuts mRNA表達(dá)量、PNUTS濃度、PⅠCP、PⅢNP濃度、膠原容積分?jǐn)?shù)及細(xì)胞凋亡率之間的相互關(guān)系。[結(jié)果]1、與對照組比較,各缺氧組大鼠血清和心肌組織中PⅠCP、PⅢNP含量顯著增加(P＜0.01);各缺氧組間兩兩分析比較顯示,血清及心肌組織中的PⅠCP、PⅢNP含量差異均有顯著性(P＜0.01),并隨著缺氧時間的增加而增加。2、與對照組比較,HE染色顯示各缺氧組大鼠心肌細(xì)胞均有不同程度的水腫、空泡變性、肥大、炎癥細(xì)胞浸潤及纖維化;Masson染色顯示心肌細(xì)胞間隙膠原蛋白增加,膠原沉積增加,缺氧組的CVF顯著增多(P0.01);病理切片熒光Tunel顯示心肌細(xì)胞凋亡增加,心肌細(xì)胞凋亡率顯著增加(P0.01)。各缺氧組間兩兩分析比較顯示:心肌病理改變、纖維化程度、CVF、心肌細(xì)胞凋亡率差異有顯著性(P＜0.01),并且隨著時間的增加而增加。3、與對照組比較,各缺氧組心肌組織miR-34a表達(dá)量顯著增加(P0.01),pnuts mRNA表達(dá)量顯著減少(P0.01);各缺氧組間兩兩比較顯示:miR-34a表達(dá)量差異顯著(P＜0.01),且隨著缺氧時間的增加而增加,pnuts mRNA表達(dá)量差異顯著(P＜0.01),且隨著缺氧時間的增加而減少。4、相關(guān)性分析顯示:大鼠血清和心肌組織的PⅠCP、PⅢNP濃度與CVF呈顯著正相關(guān)(P0.01) ; PⅠCP、PⅢNP濃度與心肌細(xì)胞凋亡率凋亡呈顯著正相關(guān)(P＜0.01)。5、干細(xì)胞中miR-34a表達(dá)量顯著低于對照組(P0.01),干細(xì)胞中pnutsmRNA表達(dá)量、PNUTS濃度顯著高于對照組(P＜0.01)。6、干細(xì)胞治療組(B'組、C'組、D'組、E'組)血清及心肌組織中的PⅠCP、PⅢNP濃度較相應(yīng)的各缺氧組顯著下降(P0.01)。7、干細(xì)胞治療組(B'組、C'組、D'組、E'組)的心肌組織異常病理改變、纖維化程度較相應(yīng)的各缺氧組顯著改善,心肌組織中的CVF及心肌細(xì)胞凋亡率較相應(yīng)的各缺氧組顯著下降(P0.01)。8、干細(xì)胞治療組(B'組、C'組、D'組)心肌組織miR-34a表達(dá)量較相應(yīng)的各缺氧組顯著減少(P0.01),pnuts mRNA表達(dá)量顯著增多(P＜0.01),PNUTS濃度顯著增多(P0.01);干細(xì)胞治療組E'組心肌組織miR-34a表達(dá)量、pnuts mRNA表達(dá)量及PNUTS濃度較缺氧組E組改變不顯著(P0.01)。9、相關(guān)性分析顯示:除E組(缺氧28天組),余各缺氧組和治療組心肌組織miR-34a表達(dá)量與pnuts mRNA表達(dá)量呈顯著負(fù)相關(guān)(P0.01),缺氧組及治療組PNUTS的含量與PⅠCP、PⅢNP含量、CVF、細(xì)胞凋亡率呈顯著負(fù)相關(guān)(P0.01)。線性回歸分析提示:缺氧組及治療組miR-34a與PNUTS含量顯著相關(guān)(R2=0.856、R2=0.862),回歸系數(shù)分別為-0.132 (P＜0.05) ; -0.162 (P0.05);PNUTS含量與缺氧組PⅠCP、PⅢNP含量顯著相關(guān)(R2=0.986、R2=0.949)回歸系數(shù)分別為-22.518(P0.05)、-11.188(P0.05);PNUTS含量與治療組PⅠCP、PⅢNP含量顯著相關(guān)(R2=0.992、R2=0.981)回歸系數(shù)分別為-7.673 (P＜0.05)、-5.478 (P0.05) ; PNUTS含量與缺氧組及治療組CVF顯著相關(guān)(R2=0.995、R2=0.992)回歸系數(shù)分別為-0.168 (P0.05)、-0.090 (P0.05) ; PNUTS 含量與缺氧組及治療組心肌細(xì)胞凋亡率顯著相關(guān)(R2=0.999、R2=0.997)回歸系數(shù)分別為-0.643 (P＜0.05)、-0.113 (P0.05)。[結(jié)論]1、慢性缺氧心室重構(gòu)模型建立成功,在缺氧21天到缺氧28天時心室發(fā)生重構(gòu)最嚴(yán)重;2、慢性缺氧心室重構(gòu)過程中,心肌纖維化、心肌細(xì)胞凋亡出現(xiàn)時間基本一致,兩者共同參與心室重構(gòu)的發(fā)展過程,且在缺氧21天到缺氧28天時改變更明顯;3、BMSCs對慢性缺氧大鼠的心室重構(gòu)有明顯改善作用,但不能完全逆轉(zhuǎn)心室重構(gòu)。4、miR-34a可以抑制pnuts在心肌中的表達(dá);5、miR-34a在大鼠骨髓間充質(zhì)干細(xì)胞中低表達(dá),pnuts在大鼠骨髓間充質(zhì)干細(xì)胞中高表達(dá);6、PNUTS與慢性缺氧導(dǎo)致的心室重構(gòu)具有顯著相關(guān)性;7、BMSCs可通過miR-34a調(diào)控的PNUTS改善慢性缺氧導(dǎo)致的心室重構(gòu),但是對于缺氧時間較長的慢性缺氧模型,BMSCs并不通過miR-34a調(diào)控的PNUTS發(fā)揮作用。
[Abstract]:[Objective] chronic hypoxia is one of the main factors leading to ventricular remodeling. Ventricular remodeling is an important pathological mechanism that causes heart failure and arrhythmia in all kinds of heart diseases. It is also a sign of heart aging. In recent years, domestic and foreign studies have found that BMSCs (bone marrow mesenchymal stem cells) transplantation can improve the heart effectively. Myocyte apoptosis and myocardial fibrosis, BMSCs transplantation is expected to be one of the feasible treatment methods to improve ventricular remodeling. However, the mechanism of BMSCs improvement of ventricular remodeling is not clear.PNUTS (Serine/threonine protein phosphatase 1 regulation 10; PPPIR10; serine / threonine protein phosphatase 1 regulation subunit 10) is protein phosphorus The regulatory subunit of acid enzyme 1 (PP1), which mainly has anti apoptosis and promotes cardiac function recovery after acute myocardial infarction, has recently shown that PNUTS can be expressed in BMSCs, but the correlation of BMSCs through PNUTS to improve ventricular remodeling caused by chronic hypoxia and the correlation of ventricular remodeling caused by PNUTS and slow cardiac hypoxia has not been reported. The chronic hypoxic ventricular remodeling model of SD rats was established and the model of hypoxia rats was treated with BMSCs. The chronic hypoxia ventricular remodeling model and the ventricular miR-34a, pnuts mRNA expression, PNUTS concentration, and P I CP, P III NP concentration in serum and myocardial tissues after BMSCs treatment were detected. The ventricular remodeling caused by chronic hypoxia was discussed. And BMSCs to improve the role of BMSCs in ventricular remodeling. [method]45 rats were randomly divided into 9 groups, 5 rats in each group, group A (control group), group B (7 days of hypoxia), group C (14 days of hypoxia), group D (21 days of hypoxia), group E (28 days of hypoxia), B'group (7 days after 7 days of hypoxia, 5 x 106/mL 1mL, 4 weeks), C' group 14 days (14 days of hypoxia) The posterior intraperitoneal injection of 1mL, 5 x 106/mL, was treated for 4 weeks. In group D'(21 days after hypoxia, 5 x 106/mL of stem cells 1mL, 4 weeks of treatment), E' group (28 days after hypoxia, 5 * 106/mL of stem cell 1mL, for 4 weeks), an anoxic group model was established with an automatic anoxic experimental device for 4 weeks. After being weighed, the rats were injected into 10% hydrochloral anaesthetized rats by intraperitoneal injection of 0.3mL/100g and dissected, open the abdominal cavity, take the blood from the abdominal aorta, and take the serum after centrifugation; open the chest and take the heart to make pathological sections. HE staining was used to observe the morphology, edema, hypertrophy, degeneration, necrosis and inflammation of the group of rats in the anoxic group and the treatment group. Pathological changes such as cell infiltration, Masson staining was used to observe the distribution and content of myocardial collagen in the hypoxia group and the treatment group, and calculate the collagen volume fraction (CVF); fluorescence Tunel was used to detect the apoptosis of cardiac muscle cells in the myocardial tissue of each anoxic group and the treatment group, and the apoptosis rate was calculated. QPCR was used to detect the hypoxia group. The concentration of P I CP, P III NP, P I CP, P III concentration, concentration, concentration, concentration, concentration, concentration, concentration, concentration, concentration, concentration, concentration, concentration, concentration, concentration, concentration of pnuts mRNA in the rats' myocardial tissue and the expression of pnuts mRNA in the BMSCs and the serum of the rats in the hypoxia group and the treatment group were detected by ELISA. The correlation between the number of integral and the rate of apoptosis. [results]1, compared with the control group, the content of P I CP and P III NP in the serum and myocardial tissues of the rats of each anoxic group increased significantly (P < 0.01). The differences in the content of P I CP and P III NP in the serum and myocardial tissues were statistically significant (P < 0.01), and with the anoxia. Increased.2, compared with the control group, HE staining showed that the myocardial cells in the hypoxia group had edema, vacuolar degeneration, hypertrophy, inflammatory cell infiltration and fibrosis; Masson staining showed the increase of collagen in the intercellular space, the increase of collagen deposition, and the increase of CVF in the hypoxia group (P0.01); pathological section fluorescence Tunel. The apoptosis rate of cardiac myocytes increased significantly (P0.01). The 22 analysis and comparison between the anoxic groups showed that the myocardial pathological changes, the degree of fibrosis, CVF, the apoptosis rate of myocardial cells were significantly different (P < 0.01), and.3 was added with the increase of time, and compared with the control group, the miR-34a expression of myocardial tissue in each anoxic group was compared with the control group. Significantly increased (P0.01) and pnuts mRNA expression decreased significantly (P0.01). The 22 comparison between the anoxic groups showed that the expression of miR-34a was significantly different (P < 0.01), and increased with the increase of the hypoxia time, and the expression of pnuts mRNA was significantly different (P < 0.01), and decreased with the increase of oxygen deficiency time. The correlation analysis showed that the serum and myocardium of rats were serum and myocardium. The concentration of P I CP and P III NP was significantly positively correlated with CVF (P0.01); P I CP, P III NP concentration was significantly positively correlated with apoptosis rate of cardiomyocytes (P < 0.01), and the expression amount in stem cells was significantly lower than that of the control group. The concentration of P I CP in serum and myocardial tissue in group C', group D' and E'group was significantly lower than that of the corresponding hypoxia group (P0.01).7. The myocardial tissue of the stem cell therapy group (B' group, C'group, D' group, group) was abnormal pathological changes, and the degree of fibrosis in the myocardial tissue was better than that of the corresponding hypoxia group. The hypoxia group decreased significantly (P0.01).8. The expression of miR-34a in myocardial tissue in the stem cell therapy group (group B', C' group, D'group) decreased significantly (P0.01), the mRNA expression of pnuts increased significantly (P < 0.01), PNUTS concentration increased significantly (P0.01). Compared with the hypoxia group, the changes in the E group were not significant (P0.01).9. The correlation analysis showed that the miR-34a expression of myocardial tissue in the hypoxia group and the treatment group was negatively correlated with the pnuts mRNA expression except the E group (28 days of hypoxia), and the content of PNUTS in the hypoxia group and the treatment group and P I CP, P III content, and the apoptosis rate showed significant negative correlation. The regression analysis showed that the content of miR-34a and PNUTS in the anoxic group and the treatment group were significantly correlated (R2=0.856, R2=0.862), the regression coefficients were -0.132 (P < 0.05), -0.162 (P0.05), PNUTS content and P I CP in the hypoxia group, and the regression coefficient of P III, respectively, and the treatment group. P I CP, P III NP content significantly correlated (R2=0.992, R2=0.981) regression coefficients were -7.673 (P < 0.05), -5.478 (P0.05), PNUTS content was significantly correlated with the hypoxia group and the treatment group. The regression coefficients of 2=0.999, R2=0.997) were -0.643 (P < 0.05) and -0.113 (P0.05). [conclusion]1, chronic hypoxia ventricular remodeling model was established successfully. The most serious remodeling of ventricle occurred during hypoxia 21 days to 28 days of hypoxia; 2. During the process of chronic hypoxia ventricular remodeling, myocardial fibrosis and myocardial cell apoptosis were basically the same, and both were involved in the heart. The development of ventricular remodeling is more obvious in 21 days of hypoxia to 28 days of hypoxia; 3, BMSCs can improve ventricular remodeling in rats with chronic hypoxia, but it can not completely reverse the ventricular remodeling.4, miR-34a can inhibit the expression of pnuts in the myocardium; 5, miR-34a is low expression in rat bone marrow mesenchymal stem cells, and pnuts in rat bone marrow High expression in mesenchymal stem cells; 6, PNUTS has a significant correlation with chronic hypoxia induced ventricular remodeling; 7, BMSCs can improve ventricular remodeling induced by chronic hypoxia by miR-34a regulated PNUTS, but BMSCs does not play a role in miR-34a regulated PNUTS in chronic hypoxia model with longer hypoxia.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R54

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